Varioskan flash spectral scanning multimode microplate reader 183

Cytochrome c oxidase (COX) activity was measured according to [75 (link)] using a microplate combining 5 μL of supernatant, 192 μL of K phosphate buffer (50 mM, 1 mM EDTA, pH 7.2), and 3 μL of 2 mM reduced cytochrome c (c2037; Sigma-Aldrich; USA; reduced with ascorbic acid 20:1) to initiate the reaction. The COX activity was measured in duplicate at 37 °C, with an absorbance of 550 nm using a microplate reader (Varioskan Flash-Spectral Scanning Multimode Microplate Reader 183, Thermo Scientific, MA, USA). The results were expressed as nmol/min/mg of protein. Protein concentration was measured with the Bradford method.

Citrate synthase (CS) activity was determined according to Dzik and coworkers [43 (link)]. In brief, 3 μL of the supernatant was combined with 181 μL of buffer (50 mM TRIS-HCl with 5 mM EDTA, pH 8.1), 20 μL of freshly made DTNB (1 mM), 2 μL of acetyl-coenzyme A (15 mM), and 2 μL of freshly made oxaloacetate acid (10 mM) to initiate the reaction. The results were obtained in 2 min by measuring the absorbance change at 412 nm using a multimode microplate reader (Varioskan Flash-Spectral Scanning Multimode Microplate Reader 183, Thermo Fisher Scientific, MA, USA) at 37 °C. The CS activity was measured in duplicate and expressed as µmol/min/mg of protein.